Preparation method and application of modified pueraria polysaccharide

Abstract

The application discloses a preparation method of modified puerariae radix polysaccharide and application thereof, and relates to the technical field of modification of natural traditional Chinese medicines. The application provides a preparation method of modified puerariae radix polysaccharide, and a method for preparing a natural puerariae radix polysaccharide conductive heart patch imitating tree frog feet and a puerariae radix polysaccharide double-crosslinked blood vessel stent coating by using the modified puerariae radix polysaccharide. The application also provides application of the modified puerariae radix polysaccharide in preparation of a medicine for treating cardiovascular diseases. The method provided by the application has high modification efficiency and stable modification effect, and effectively solves the problems of low modification efficiency, many by-products and poor solubility of puerariae radix polysaccharide in the prior art.

Description

Technical Field

[0001] This invention relates to the field of modification technology of natural Chinese medicine, specifically to a method for preparing modified kudzu polysaccharide and its application. Background Technology

[0002] Kudzu root, a natural Chinese medicinal herb, is rich in various isoflavones, accounting for approximately 12% of its composition, with puerarin being the main component. Studies have shown that puerarin possesses multiple effects, including reversing lipid peroxidation damage, promoting angiogenesis, and antibacterial properties. However, kudzu polysaccharides have poor natural solubility, making them difficult to dissolve rapidly at room temperature, and the resulting solutions have high viscosity, limiting their application in biomaterials.

[0003] In existing technologies, the double bond conversion strategy for starch-based materials often involves direct modification with glycidyl methacrylate esters. However, this modification method is inefficient and produces numerous byproducts. Furthermore, traditional methods fail to effectively introduce active groups, resulting in materials lacking tissue adhesion or dynamic cross-linking capabilities. Due to the poor solubility of kudzu raw materials and the lack of chemically active sites, its application range is limited.

[0004] Furthermore, while hydrogel materials constructed from commonly used natural polymers and their derivatives exhibit good biocompatibility in the treatment of cardiovascular diseases, the limited functional modules significantly restrict their ability to synergistically regulate the pathological microenvironment. Specifically, gelatin-based polymers lack endogenous reactive oxygen species scavenging groups, such as phenolic hydroxyl and thiol groups, and chitosan-based materials have low conductivity rates in their insulating matrix, hindering the synchronicity of transmembrane calcium cis-transformation. These shortcomings have constrained the clinical translation of hydrogel materials. Summary of the Invention

[0005] In view of the above-mentioned shortcomings in the prior art, the present invention provides a method for preparing modified kudzu polysaccharide and its application, which effectively solves the problems of low modification efficiency, many by-products and poor solubility of kudzu polysaccharide in the prior art.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is: to provide a modified kudzu polysaccharide, including oxidized kudzu polysaccharide, double-bonded kudzu polysaccharide and thiolated kudzu polysaccharide.

[0007] The above-mentioned method for preparing oxidized kudzu polysaccharide includes the following steps: dissolving kudzu polysaccharide in reverse osmosis water, then adding boiling water, stirring and cooling, then adding sodium periodate solution dropwise, adding ethylene glycol after reaction, and finally dialyzing and freeze-drying to obtain oxidized kudzu polysaccharide.

[0008] Furthermore, the mass-to-volume ratio of kudzu polysaccharide, reverse osmosis water, sodium periodate solution, and boiling water is 7-9 g: 40-60 mL: 40-60 mL: 90-110 mL.

[0009] Furthermore, the mass-to-volume ratio of kudzu polysaccharide, reverse osmosis water, sodium periodate solution, and boiling water was 8 g:50 mL:50 mL:100 mL.

[0010] Furthermore, the concentration of the sodium periodate solution is 0.3-0.5 M.

[0011] Furthermore, the concentration of the sodium periodate solution is 0.4 M.

[0012] Further, stir until the solution turns brown, then cool to 35-45 ℃.

[0013] Further, stir until the solution turns brown, then cool to 40 °C.

[0014] Further, add sodium periodate solution dropwise over 25-35 minutes.

[0015] Further, sodium periodate solution was added dropwise over 30 minutes.

[0016] Furthermore, the reaction was carried out in a water bath at 35-45 ℃ in the dark for 4-5 hours.

[0017] Furthermore, the reaction was carried out in a 40 ℃ water bath in the dark for 4.5 h.

[0018] Furthermore, the mass-to-volume ratio of kudzu polysaccharide to ethylene glycol is 7-9 g: 2-3 mL.

[0019] Furthermore, the mass-to-volume ratio of kudzu polysaccharide to ethylene glycol is 8 g: 2.1 mL.

[0020] Furthermore, dialysis was performed for 6–8 days in a dialysis bag with a capacity of 3500 Da.

[0021] Furthermore, dialysis was performed for 7 days in a dialysis bag with a capacity of 3500 Da.

[0022] Furthermore, it is freeze-dried under vacuum at -50℃ for 70-80 hours.

[0023] Furthermore, it was freeze-dried under vacuum at -50°C for 72 hours.

[0024] The above-mentioned method for preparing double-bonded kudzu polysaccharide includes the following steps: carboxymethylating kudzu polysaccharide in an alkaline environment, drying to obtain carboxymethylated kudzu polysaccharide, then dissolving it in reverse osmosis water, adjusting the pH after heating, adding glycidyl methacrylate to react, cooling, dialyzing and freeze-drying to obtain double-bonded kudzu polysaccharide.

[0025] Further, the carboxymethylation of kudzu polysaccharide includes the following steps: 140-160 mL of 80-90 vt % ethanol solution is added to 10-11 g of sodium hydroxide, followed by 16-17 g of kudzu polysaccharide. The mixture is heated in a constant temperature water bath at 25-35 ℃ for alkalization for 1-2 h, then 16-18 g of chloroacetic acid is added, and the temperature is raised to 35-45 ℃ for etherification for 3-5 h. The filtrate is filtered off, and the mixture is vacuum dried at 50-60 ℃ for 20-30 h. Then, 75-85 vt % ethanol is added, and the pH is adjusted to neutral with 1-3 mol / L hydrochloric acid solution. After filtration, the mixture is washed 4-5 times with 75-85 vt % ethanol solution, and then dried to obtain carboxymethylated kudzu polysaccharide.

[0026] Further, the carboxymethylation of kudzu polysaccharide includes the following steps: 150 mL of 85 vt % ethanol solution is added to 10.8 g of sodium hydroxide, followed by the addition of 16.2 g of kudzu polysaccharide. The mixture is heated in a constant temperature water bath at 30 ℃ for alkalization for 1.5 h, then 17 g of chloroacetic acid is added, and the mixture is heated to 40 ℃ for etherification for 4 h. The filtrate is filtered off, and the mixture is vacuum dried at 55 ℃ for 24 h. The mixture is then added to 80 vt % ethanol, and the pH is adjusted to neutral with 2 mol / L hydrochloric acid solution. After filtration, the mixture is washed 5 times with 80 vt % ethanol solution and dried to obtain carboxymethylated kudzu polysaccharide.

[0027] Furthermore, the mass-to-volume ratio of carboxymethylated kudzu polysaccharide to reverse osmosis water is 1-3 g: 150-250 mL.

[0028] Furthermore, the mass-to-volume ratio of carboxymethylated kudzu polysaccharide to reverse osmosis water was 2 g: 200 mL.

[0029] Further, after heating the water bath to 85-95 ℃, add hydrochloric acid to adjust the pH to 3-4.

[0030] Furthermore, after heating the solution to 90 °C in a water bath, hydrochloric acid was added to adjust the pH to 3.5.

[0031] Furthermore, the mass-to-volume ratio of carboxymethylated kudzu polysaccharide to glycidyl methacrylate is 1-3 g: 4-6 mL.

[0032] Furthermore, the mass-to-volume ratio of carboxymethylated kudzu polysaccharide to glycidyl methacrylate is 2 g: 5 mL.

[0033] Furthermore, the reaction takes 6-10 hours.

[0034] Furthermore, dialysis was performed for 7 days after natural cooling.

[0035] The above-mentioned method for preparing thiolated kudzu polysaccharide includes the following steps: carboxymethylating kudzu polysaccharide in an alkaline environment to obtain carboxymethylated kudzu polysaccharide, then adding reverse osmosis water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide and stirring, then adding thioglycolic acid, stirring and reacting, dialyzing and freeze-drying to obtain thiolated kudzu polysaccharide.

[0036] Furthermore, the mass-to-volume ratio of carboxymethylated kudzu polysaccharide, N-hydroxysuccinimide, reverse osmosis water, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1-3 g: 450-550 mg: 450-550 mg: 150-250 mL.

[0037] Furthermore, the mass-to-volume ratio of carboxymethylated kudzu polysaccharide, N-hydroxysuccinimide, reverse osmosis water, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was 2 g:500 mg:500 mg:200 mL.

[0038] Continue stirring for 1-3 hours.

[0039] Continue stirring for 2 hours.

[0040] Furthermore, the mass ratio of thioglycolic acid to carboxymethylated kudzu polysaccharide is 1-3 g: 1-3 g.

[0041] Furthermore, the mass ratio of thioglycolic acid to carboxymethylated kudzu polysaccharide is 2 g: 2 g.

[0042] Furthermore, the mixture was stirred at room temperature and reacted in the dark for 23-25 ​​hours.

[0043] Furthermore, the mixture was stirred at room temperature and reacted in the dark for 24 hours.

[0044] Furthermore, the cells were freeze-dried 7 days after dialysis.

[0045] Modified kudzu polysaccharide was prepared using the above-described method.

[0046] A conductive cardiac patch incorporating natural kudzu polysaccharide mimicking the foot of a tree frog, comprising the aforementioned oxidized kudzu polysaccharide.

[0047] The preparation method of the above-mentioned tree frog-inspired natural kudzu polysaccharide conductive cardiac patch includes the following steps: S1. Heat and stir the tetrachloroauric acid trihydrate solution to boiling, add sodium citrate solution, and after the reaction, gold nanoparticles are obtained. S2. The gold nanoparticles obtained in step S1 were modified with cysteine ​​using the Liang method, stirred and washed, and then dispersed in ultrapure water to obtain Cys @ AuNPs; S3. Construct a 3D mold of the tree frog's foot. Dissolve the above-mentioned oxidized kudzu polysaccharide and gel in reverse osmosis water, and then mix it evenly with the Cys @ AuNPs obtained in step S2 to obtain a mixed solution. Coat the mixed solution into the 3D mold of the tree frog's foot. After gelation, obtain a conductive heart patch imitating the natural kudzu polysaccharide of the tree frog's foot.

[0048] Furthermore, in step S1, the mass-to-volume ratio of tetrachloroauric acid trihydrate solution to sodium citrate is 45-55 mL: 0.5-1.5 mL.

[0049] Furthermore, in step S1, the volume ratio of tetrachloroauric acid trihydrate solution to sodium citrate is 50 mL: 1 mL.

[0050] Furthermore, in step S1, the concentration of the tetrachloroauric acid trihydrate solution is 0.2-0.4 mmol / L.

[0051] Furthermore, in step S1, the concentration of the tetrachloroauric acid trihydrate solution is 0.3 mmol / L.

[0052] Furthermore, in step S1, the concentration of the sodium citrate solution is 38-39 mmol / L.

[0053] Furthermore, in step S1, the concentration of the sodium citrate solution is 38.7 mmol / L.

[0054] Furthermore, in step S1, the reaction continues for 5-6 minutes until the solution turns red.

[0055] Furthermore, in step S1, the reaction continues for 5 minutes until the solution turns red.

[0056] Furthermore, in step S2, the ratio of cysteine ​​to gold nanoparticles is 49-51:1.

[0057] Furthermore, in step S2, the ratio of cysteine ​​to gold nanoparticles is 50:1.

[0058] Furthermore, in step S2, the reaction is stirred for 23-25 ​​hours.

[0059] Furthermore, in step S2, the reaction is stirred for 24 hours.

[0060] Furthermore, in step S2, the centrifugation and washing are performed 3-4 times.

[0061] Furthermore, in step S2, the centrifugation and washing are performed three times.

[0062] Furthermore, in step S2, the concentration of gold nanoparticles in Cys@AuNPs is 0.01-1 mg / mL.

[0063] Furthermore, in step S2, the concentration of gold nanoparticles in Cys@AuNPs is 0.05 mg / mL.

[0064] Furthermore, in step S3, a 3D mold of the tree frog's legs is constructed using SolidWorks.

[0065] Furthermore, in step S3, the tree frog foot 3D mold has a regular hexagonal honeycomb structure with a side length of 550-650 μm, a thickness of 250-350 μm, and a spacing of 50-150 μm.

[0066] Furthermore, in step S3, the tree frog foot 3D mold has a regular hexagonal honeycomb structure with a side length of 600 μm, a thickness of 300 μm, and a spacing of 100 μm.

[0067] Furthermore, in step S3, a biocompatible resin is used to construct the layer using a 3D printer (microarchs140pro, SLA) with a printing accuracy of 9-11 μm, an exposure time of 4500-5500 ms, and a single layer thickness of 0.01-0.03 mm.

[0068] Furthermore, in step S3, a biocompatible resin is used to construct the layer using a 3D printer (microarchs140pro, SLA) with a printing accuracy of 10 μm, an exposure time of 5000 ms, and a single layer thickness of 0.02 mm.

[0069] Furthermore, in step S3, the concentration of oxidized kudzu polysaccharide in the mixed solution is 9-11 wt%.

[0070] Furthermore, in step S3, the concentration of oxidized kudzu polysaccharide in the mixed solution is 10 wt%.

[0071] Furthermore, in step S3, the concentration of the gel in the mixed solution is 14-16 wt%.

[0072] Furthermore, in step S3, the concentration of the gel in the mixed solution is 15 wt%.

[0073] Furthermore, in step S3, the gel is type A porcine skin gelatin.

[0074] Furthermore, in step S3, the concentration of Cys@AuNPs in the mixed solution is 0.01-0.1 mg / mL. Furthermore, in step S3, the concentration of Cys@AuNPs in the mixed solution is 0.05 mg / mL.

[0075] Furthermore, in step S3, after gelation at 36-38℃, the gel is cooled to 3-5℃ and removed.

[0076] Furthermore, in step S3, the gel is formed at 37°C and then cooled to 4°C before being removed.

[0077] A kudzu polysaccharide double crosslinked vascular stent coating comprising double-bonded polysaccharides and thiolated kudzu polysaccharides.

[0078] The preparation method of the above-mentioned kudzu polysaccharide double cross-linked vascular stent coating includes the following steps: (1) Clean the stainless steel substrate, then perform ion beam assisted plasma treatment, clean and dry to obtain plasma-modified stainless steel; (2) The above double-bonded kudzu polysaccharide and thiolized kudzu polysaccharide are dissolved in reverse osmosis water to obtain a mixed solution. Then, the plasma-modified stainless steel obtained in step S1 is immersed in the mixed solution. After the reaction, it is washed and dried to obtain a kudzu polysaccharide double crosslinked vascular stent coating.

[0079] Furthermore, in step (1), the stainless steel substrate has dimensions of 9-11 cm * 9-11 cm * 0.1-0.3 cm.

[0080] Furthermore, in step (1), the stainless steel substrate has dimensions of 10 cm * 10 cm * 0.2 cm.

[0081] Furthermore, in step (1), the sample is ultrasonically cleaned in acetone and ethanol for 18-22 min at 24-26 °C.

[0082] Furthermore, in step (1), the sample is ultrasonically cleaned in acetone and ethanol for 20 min each at 25 °C.

[0083] Furthermore, in step (1), after cleaning, rinse with ultrapure water.

[0084] Further, in step (1), the ion beam-assisted plasma treatment includes the following steps: fixing the stainless steel substrate on the sample holder, connecting the cylindrical ion-assisted plasma deposition device (IAPP) to the radio frequency power supply (ENI RF), connecting the substrate support to the RUP-6 pulse generator (GBS Elektronik GmbH), enhancing the ion bombardment effect by applying a negative bias voltage, the vacuum system of the IAPP device consists of an Ebara PDV250 mechanical pump and an Edwards NEXT400 turbomolecular pump, connected to the chamber through a butterfly valve, introducing the precursor gas into the system through a shower head device, connecting the IAPP device to the Allicat Scientific mass flow controller, electrically connecting the sample holder to the grid 5 cm in front of it, and after cleaning, evacuating the chamber to 4 × 10⁻⁶. -5 -6×10 -5Torr is then introduced, followed by the introduction of thin film deposition gas. Acetylene, nitrogen, and argon are mixed and introduced into the system at constant flow rates of 4-6 sccm, 4-6 sccm, and 19-21 sccm, respectively. After adjusting the chamber working pressure to 100-120 mTorr, 45-55 W of radio frequency power and 450-550 V of negative bias are applied, and deposition continues for 3-5 min.

[0085] Further, in step (1), the ion beam-assisted plasma treatment includes the following steps: fixing the stainless steel substrate on the sample holder, connecting the cylindrical ion-assisted plasma deposition device (IAPP) to the radio frequency power supply (ENI RF), connecting the substrate support to the RUP-6 pulse generator (GBS Elektronik GmbH), enhancing the ion bombardment effect by applying a negative bias voltage, the vacuum system of the IAPP device consists of an Ebara PDV250 mechanical pump and an Edwards NEXT400 turbomolecular pump, connected to the chamber through a butterfly valve, introducing the precursor gas into the system through a shower head device, connecting the IAPP device to the Allicat Scientific mass flow controller, electrically connecting the sample holder to the grid 5 cm in front of it, and after cleaning, evacuating the chamber to 5 × 10⁻⁶. -5 Torr was then introduced, followed by the introduction of thin film deposition gas. Acetylene, nitrogen, and argon were mixed and introduced into the system at constant flow rates of 5 sccm, 5 sccm, and 20 sccm, respectively. After adjusting the working pressure in the chamber to 110 mTorr, 50W of radio frequency power and 500V negative bias were applied, and deposition continued for 4 min.

[0086] Furthermore, in step (1), the ion-assisted plasma deposition device is made of stainless steel.

[0087] Furthermore, in step (1), the frequency of the radio frequency power supply is 13-14 MHz.

[0088] Furthermore, in step (1), the frequency of the radio frequency power supply is 13.56 MHz.

[0089] Furthermore, in step (1), the product is ultrasonically cleaned in ethanol and single-distilled water in sequence and then dried in a vacuum drying oven at 36-38 ℃ for 2-3 days.

[0090] Furthermore, in step (1), the product is ultrasonically cleaned in ethanol and single-distilled water in sequence and then dried in a vacuum drying oven at 37 °C for 2 days.

[0091] Furthermore, in step (2), the concentration of double-bonded kudzu polysaccharide in the mixed solution is 4-6 wt%.

[0092] Furthermore, in step (2), the concentration of double-bonded kudzu polysaccharide in the mixed solution is 5 wt%.

[0093] Furthermore, in step (2), the concentration of thiolated kudzu polysaccharide in the mixed solution is 4-6 wt%.

[0094] Furthermore, in step (2), the concentration of thiolated kudzu polysaccharide in the mixed solution is 5 wt%.

[0095] Furthermore, in step (2), after reacting at 36-38℃ for 23-25 ​​h, the mixture is washed with single-distilled water and dried with nitrogen.

[0096] Furthermore, in step (2), after reacting at 37°C for 24 h, the mixture is washed with single-distilled water and dried with nitrogen.

[0097] The above-mentioned modified kudzu polysaccharide, tree frog-inspired natural kudzu polysaccharide conductive cardiac patch, and kudzu polysaccharide double cross-linked vascular stent coating are used in the preparation of drugs for treating cardiovascular diseases.

[0098] In summary, the present invention has the following beneficial effects: 1. The double bond modification method of kudzu polysaccharide provided by this invention is efficient and rapid, and contains no toxic raw materials. It reacts in mild water, has a high yield, and imparts rapid photocuring properties to kudzu polysaccharide (5% concentration UV-responsive curing time is about 1 second). The solubility in water is greatly improved compared to the solubility of the original raw materials and carboxymethylated materials. This property enables it to be applied in a variety of hydrogel systems and form a tight cross-linked network, effectively solving the problems of kudzu polysaccharide's lack of responsiveness and poor solubility.

[0099] 2. Oxidized kudzu polysaccharide imparts carboxyl active groups to the material and greatly improves the solubility of kudzu polysaccharide, enabling it to dissolve rapidly at room temperature with extremely low solution viscosity and a high rate of active group modification.

[0100] 3. Thiolization modification endows kudzu polysaccharide with good tissue adhesion ability and has click chemically active groups, which can be widely used in free radical polymerization hydrogel systems to provide good component support.

[0101] 4. Kudzu polysaccharide is derived from the raw material of kudzu root powder, a traditional Chinese medicine. It is rich in a large number of effective medicinal components. However, its inherent characteristics greatly limit its application in many fields. This invention modifies kudzu polysaccharide to retain its medicinal properties to the greatest extent and at the same time give it a better application mode to realize its application in multiple application scenarios, especially in the pathological model of cardiovascular diseases.

[0102] 5. The tree frog-foot-inspired conductive cardiac patch provided by this invention is 3D printed in a honeycomb shape. It achieves a dynamic contraction tensile strength of 2292 kPa and an elongation of 650% when applied to the heart. It can promote the expression of CX43 protein in cardiomyocytes, improve electrical signal synchronization, and effectively inhibit Staphylococcus aureus. It also restores the ejection fraction of mice with myocardial infarction to 73.7% and reduces the fibrosis area to 18.75%. In the double-crosslinked vascular stent coating of kudzu polysaccharide, the double-bonded and thiolized kudzu polysaccharides work synergistically to enhance the stability of the coating. Attached Figure Description

[0103] Figure 1 Rheological properties of different patches; Figure 2 To assess the healing ability of the patch under different shear stresses; Figure 3 Tensile stress-strain curves for different patches; Figure 4 Figure showing the results of the anti-Gram-positive bacteria performance of different patches; Figure 5 Fluorescence image of CX43 protein expression in cardiomyocytes; Figure 6 The image shows the semi-quantitative fluorescence analysis results of CX43. Figure 7 30D live cardiac image; Figure 8 Image of mouse heart tissue stained with WGA fluorescence. Figure 9 A graph showing the proportion of myocardial infarction in mice and a comparison of the cross-sectional area of ​​the distal infarct region in different groups; Figure 10 SEM image of the PSMASH surface; Figure 11 CLSM images with different layers. Detailed Implementation

[0104] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0105] Example 1 The preparation method of oxidized kudzu polysaccharide includes the following steps: 8 g of kudzu polysaccharide is dissolved in 50 mL of reverse osmosis water, then 100 mL of boiling water is added, and the mixture is stirred until the solution turns brown. The solution is then cooled to 40 °C, and 50 mL of 0.4 M sodium periodate solution is added dropwise over 30 min. The mixture is then reacted in a 40 °C water bath in the dark for 4.5 h. 2.1 mL of ethylene glycol is added, and the mixture is finally dialyzed in a 3500 Da dialysis bag for 7 days and then freeze-dried under vacuum at -50 °C for 72 h to obtain oxidized kudzu polysaccharide.

[0106] The preparation method of double-bonded kudzu polysaccharide includes the following steps: Carboxymethylation of kudzu polysaccharide in an alkaline environment: 150 mL of 85 vt % ethanol solution is added to 10.8 g of sodium hydroxide, followed by the addition of 16.2 g of kudzu polysaccharide. The mixture is heated in a constant temperature water bath at 30 ℃ for 1.5 h for alkalization. Then, 17 g of chloroacetic acid is added, and the temperature is raised to 40 ℃ for etherification for 4 h. The filtrate is filtered off, and the mixture is vacuum dried at 55 ℃ for 24 h. Then, 80 vt % ethanol is added, and the pH is adjusted to neutral with 2 mol / L hydrochloric acid solution. After filtration, the mixture is washed 5 times with 80 vt % ethanol solution and dried to obtain carboxymethylated kudzu polysaccharide. Then, 2 g of carboxymethylated kudzu polysaccharide is dissolved in 200 mL of reverse osmosis water, heated to 90 ℃ in a water bath, and the pH is adjusted to 3.5 with hydrochloric acid. Finally, 5 mL of glycidyl methacrylate is added and reacted for 8 hours. After natural cooling, the mixture was dialyzed for 7 days and then freeze-dried to obtain double-bonded kudzu polysaccharide.

[0107] The preparation method of thiolated kudzu polysaccharide includes the following steps: kudzu polysaccharide is carboxymethylated in an alkaline environment using the above method to obtain carboxymethylated kudzu polysaccharide; then, 200 mL of reverse osmosis water, 500 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 500 mg of N-hydroxysuccinimide are added to 2 g of carboxymethylated kudzu polysaccharide and stirred for 2 h; then, 2 g of mercaptoacetic acid is added, and the mixture is stirred at room temperature and reacted in the dark for 24 h; after dialyzing for 7 days, it is freeze-dried to obtain thiolated kudzu polysaccharide.

[0108] A method for preparing a conductive cardiac patch inspired by the natural kudzu polysaccharide of the tree frog's foot, comprising the following steps: S1. Heat and stir 50 mL of 0.3 mmol / L tetrachloroauric acid trihydrate solution to boiling, add 1 mL of 38.7 mmol / L sodium citrate solution, react for 5 min until the solution turns red, and gold nanoparticles are obtained. S2. The gold nanoparticles obtained in step S1 were modified with cysteine ​​using the Liang method. The ratio of cysteine ​​to gold nanoparticles was 50:1. After stirring for 24 h, the mixture was centrifuged and washed three times, and then dispersed in ultrapure water to obtain Cys @ AuNPs. The concentration of the above gold nanoparticles in Cys @ AuNPs was 0.1 mg / mL. S3. A 3D mold of the tree frog's foot was constructed using SolidWorks. The mold had a hexagonal honeycomb structure with a side length of 600 μm, a thickness of 300 μm, and a spacing of 100 μm. The oxidized kudzu polysaccharide and type A porcine skin gelatin were dissolved in reverse osmosis water and then mixed thoroughly with the Cys@AuNPs obtained in step S2 to obtain a mixed solution. The concentration of oxidized kudzu polysaccharide in the mixed solution was 10 wt%, the concentration of type A porcine skin gelatin was 15 wt%, and the concentration of Cys@AuNPs was 0.05 mg / mL. The mixture was constructed using a 3D printer (Microarchs 140pro, SLA) with biocompatible resin, a printing precision of 10 μm, an exposure time of 5000 ms, and a single-layer thickness of 0.02 mm. The mixed solution was coated onto the tree frog's foot 3D mold and gelled at 37°C before cooling to 4°C. Removed at ℃, a conductive cardiac patch made of natural kudzu polysaccharide mimicking the foot of a tree frog was obtained.

[0109] A method for preparing a kudzu polysaccharide double crosslinked vascular stent coating includes the following steps: (1) Cleaning the stainless steel substrate (SS): The stainless steel substrate has dimensions of 10 cm * 10 cm * 0.2 cm. It is ultrasonically cleaned in acetone and ethanol for 20 min each at 25 ℃. After cleaning, it is rinsed with ultrapure water and then subjected to ion beam-assisted plasma treatment. The stainless steel substrate is fixed on the sample holder. The cylindrical ion-assisted plasma deposition device (IAPP) made of stainless steel is connected to the radio frequency power supply (ENI RF). The frequency of the radio frequency power supply is 13.56 MHz. The substrate support is connected to the RUP-6 pulse generator (GBS Elektronik GmbH). The ion bombardment effect is enhanced by applying a negative bias voltage. The vacuum system of the IAPP device consists of an Ebara PDV250 mechanical pump and an Edwards NEXT400 turbomolecular pump, which is connected to the chamber through a butterfly valve. The precursor gas is introduced into the system through a shower head device. The IAPP device is connected to the Allicat Scientific mass flow controller. The sample holder is electrically connected to the grid 5 cm in front of it. After cleaning, the chamber is evacuated to 5 × 10 -5Torr was then introduced, followed by the introduction of thin film deposition gas. Acetylene, nitrogen, and argon were mixed and introduced into the system at constant flow rates of 5 sccm, 5 sccm, and 20 sccm, respectively. After adjusting the chamber working pressure to 110 mTorr, 50 W of radio frequency power and 500 V of negative bias were applied, and deposition was carried out for 4 min. The material was then ultrasonically cleaned in ethanol and single-distilled water, and dried in a vacuum drying oven at 37 ℃ for 2 days to obtain plasma-modified stainless steel (T-SS). (2) The above-mentioned double-bonded kudzu polysaccharide and thiolated kudzu polysaccharide were dissolved in reverse osmosis water to obtain a mixed solution. The concentration of the double-bonded kudzu polysaccharide in the mixed solution was 5 wt%, and the concentration of the thiolated kudzu polysaccharide in the mixed solution was 5 wt%. Then, the plasma-modified stainless steel obtained in step S1 was immersed in the mixed solution and reacted at 37°C for 24 h. After washing with single-distilled water and drying with nitrogen, a kudzu polysaccharide double-crosslinked vascular stent coating was obtained. (PSMASH) Example 2 The preparation method of oxidized kudzu polysaccharide includes the following steps: 7g of kudzu polysaccharide is dissolved in 40 mL of reverse osmosis water, then 90 mL of boiling water is added, and the mixture is stirred until the solution turns brown. The solution is then cooled to 35 °C, and 40 mL of 0.3 M sodium periodate solution is added dropwise over 25 min. The mixture is then reacted in a water bath at 35 °C in the dark for 4 h. After that, 2 mL of ethylene glycol is added, and the mixture is dialyzed in a 3500 Da dialysis bag for 6 days and then freeze-dried under vacuum at -50 °C for 70 h to obtain oxidized kudzu polysaccharide.

[0110] The preparation method of double-bonded kudzu polysaccharide includes the following steps: Carboxymethylation of kudzu polysaccharide in an alkaline environment: 140 mL of 80 vt % ethanol solution is added to 10 g of sodium hydroxide, followed by the addition of 16 g of kudzu polysaccharide. The mixture is heated in a constant temperature water bath at 25°C for 1 h for alkalization. Then, 16 g of chloroacetic acid is added, and the temperature is raised to 35°C for etherification for 3 h. The filtrate is filtered off, and the mixture is vacuum dried at 50°C for 20 h. Then, 75 vt % ethanol is added, and the pH is adjusted to neutral with 1 mol / L hydrochloric acid solution. After filtration, the mixture is washed four times with 75 vt % ethanol solution and dried to obtain carboxymethylated kudzu polysaccharide. Then, 1 g of carboxymethylated kudzu polysaccharide is dissolved in 150 mL of reverse osmosis water, heated in a water bath to 90°C, and the pH is adjusted to 3 with hydrochloric acid. Then, 4 mL of glycidyl methacrylate is added, and the reaction is carried out for 6 h. After natural cooling, the mixture is dialyzed for 7 hours. d. The mixture was freeze-dried to obtain double-bonded kudzu polysaccharide.

[0111] The preparation method of thiolated kudzu polysaccharide includes the following steps: kudzu polysaccharide is carboxymethylated in an alkaline environment using the above method to obtain carboxymethylated kudzu polysaccharide; then, 150 mL of reverse osmosis water, 450 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 450 mg of N-hydroxysuccinimide are added to 1 g of carboxymethylated kudzu polysaccharide and stirred for 1 h; then, 1 g of mercaptoacetic acid is added, and the mixture is stirred at room temperature and reacted in the dark for 23 h; after dialyzing for 7 days, it is freeze-dried to obtain thiolated kudzu polysaccharide.

[0112] A method for preparing a conductive cardiac patch inspired by the natural kudzu polysaccharide of the tree frog's foot, comprising the following steps: S1. Heat and stir 45 mL of 0.2 mmol / L tetrachloroauric acid trihydrate solution to boiling, add 0.5 mL of 38 mmol / L sodium citrate solution, react for 5 min until the solution turns red, and gold nanoparticles are obtained. S2. The gold nanoparticles obtained in step S1 were modified with cysteine ​​using the Liang method. The ratio of cysteine ​​to gold nanoparticles was 49:1. After stirring for 23 h, the mixture was centrifuged and washed three times, and then dispersed in ultrapure water to obtain Cys @ AuNPs. The concentration of the above gold nanoparticles in Cys @ AuNPs was 0.01 mg / mL. S3. A 3D mold of the tree frog's foot was constructed using SolidWorks. The mold had a hexagonal honeycomb structure with a side length of 550 μm, a thickness of 250 μm, and a spacing of 50 μm. The oxidized kudzu polysaccharide and type A porcine skin gelatin were dissolved in reverse osmosis water and then mixed thoroughly with the Cys@AuNPs obtained in step S2 to obtain a mixed solution. The concentration of oxidized kudzu polysaccharide in the mixed solution was 9 wt%, the concentration of type A porcine skin gelatin was 14 wt%, and the concentration of Cys@AuNPs was 0.01 mg / mL. The mixture was constructed using a 3D printer (Microarchs 140 Pro, SLA) with biocompatible resin, a printing precision of 10 μm, an exposure time of 5000 ms, and a single-layer thickness of 0.02 mm. The mixed solution was coated onto the tree frog's foot 3D mold, gelled at 36°C, and then cooled to 30°C. Removed at ℃, a conductive cardiac patch made of natural kudzu polysaccharide mimicking the foot of a tree frog was obtained.

[0113] A method for preparing a kudzu polysaccharide double crosslinked vascular stent coating includes the following steps: (1) Clean the stainless steel substrate. The stainless steel sheet has a size of 9 cm*9 cm*0.1 cm. It is ultrasonically cleaned in acetone and ethanol for 18 min each at 24 ℃. After cleaning, it is rinsed with ultrapure water and then subjected to ion beam-assisted plasma treatment. The stainless steel substrate is fixed on the sample holder. The cylindrical ion-assisted plasma deposition device (IAPP) made of stainless steel is connected to the radio frequency power supply (ENI RF). The frequency of the radio frequency power supply is 13 MHz. The substrate support is connected to the RUP-6 pulse generator (GBS Elektronik GmbH). The ion bombardment effect is enhanced by applying a negative bias voltage. The vacuum system of the IAPP device consists of an Ebara PDV250 mechanical pump and an Edwards NEXT400 turbomolecular pump. It is connected to the chamber through a butterfly valve. The precursor gas is introduced into the system through a shower head device. The IAPP device is connected to the Allicat Scientific mass flow controller. The sample holder is electrically connected to the grid 5 cm in front of it. After cleaning, the chamber is evacuated to 4×10 -5 Torr was then introduced, followed by thin film deposition gas. Acetylene, nitrogen, and argon were mixed and introduced into the system at constant flow rates of 4 sccm, 4 sccm, and 19 sccm, respectively. After adjusting the working pressure of the chamber to 100 mTorr, 45W radio frequency power and 450V negative bias were applied. Deposition lasted for 3 min. The material was then ultrasonically cleaned in ethanol and single-distilled water and dried in a vacuum drying oven at 36 °C for 2 days to obtain plasma-modified stainless steel. (2) The above-mentioned double-bonded kudzu polysaccharide and thiolized kudzu polysaccharide were dissolved in reverse osmosis water to obtain a mixed solution. The concentration of double-bonded kudzu polysaccharide in the mixed solution was 4 wt%, and the concentration of thiolized kudzu polysaccharide in the mixed solution was 4 wt%. Then, the plasma-modified stainless steel obtained in step S1 was immersed in the mixed solution and reacted at 36°C for 23 h. After that, it was washed with single-distilled water and dried with nitrogen to obtain a kudzu polysaccharide double crosslinked vascular stent coating.

[0114] Example 3 The preparation method of oxidized kudzu polysaccharide includes the following steps: 9 g of kudzu polysaccharide is dissolved in 60 mL of reverse osmosis water, then 110 mL of boiling water is added, and the mixture is stirred until the solution turns brown. The solution is then cooled to 45 °C, and 60 mL of 0.5 M sodium periodate solution is added dropwise over 35 min. The mixture is then reacted in a water bath at 45 °C in the dark for 5 h, and 3 mL of ethylene glycol is added. Finally, the mixture is dialyzed in a 3500 Da dialysis bag for 8 days and then freeze-dried under vacuum at -50 °C for 80 h to obtain oxidized kudzu polysaccharide.

[0115] The preparation method of double-bonded kudzu polysaccharide includes the following steps: Carboxymethylation of kudzu polysaccharide in an alkaline environment: 160 mL of 90 vt % ethanol solution is added to 11 g of sodium hydroxide, followed by the addition of 17 g of kudzu polysaccharide. The mixture is heated in a constant temperature water bath at 35°C for 2 h for alkalization. 18 g of chloroacetic acid is then added, and the temperature is raised to 45°C for etherification for 5 h. The filtrate is filtered off, and the mixture is vacuum dried at 60°C for 30 h. Then, 85 vt % ethanol is added, and the pH is adjusted to neutral with 3 mol / L hydrochloric acid solution. After filtration, the mixture is washed 5 times with 85 vt % ethanol solution and dried to obtain carboxymethylated kudzu polysaccharide. 3 g of carboxymethylated kudzu polysaccharide is dissolved in 200 mL of reverse osmosis water, and the mixture is heated in a water bath to 90°C. Hydrochloric acid is added to adjust the pH to 3.5, followed by the addition of 5 mL of glycidyl methacrylate and reaction for 8 h. After natural cooling, the mixture is dialyzed for 7 hours. d. The mixture was freeze-dried to obtain double-bonded kudzu polysaccharide.

[0116] The preparation method of thiolated kudzu polysaccharide includes the following steps: kudzu polysaccharide is carboxymethylated in an alkaline environment using the above method to obtain carboxymethylated kudzu polysaccharide; then, 250 mL of reverse osmosis water, 550 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 550 mg of N-hydroxysuccinimide are added to 3 g of carboxymethylated kudzu polysaccharide and stirred for 3 h; then, 3 g of mercaptoacetic acid is added, and the mixture is stirred at room temperature and reacted in the dark for 25 h; after dialyzing for 7 days, it is freeze-dried to obtain thiolated kudzu polysaccharide.

[0117] A method for preparing a conductive cardiac patch inspired by the natural kudzu polysaccharide of the tree frog's foot, comprising the following steps: S1. Heat and stir 55 mL of 0.4 mmol / L tetrachloroauric acid trihydrate solution to boiling, add 1.5 mL of 39 mmol / L sodium citrate solution, react for 6 min until the solution turns red, and gold nanoparticles are obtained. S2. The gold nanoparticles obtained in step S1 were modified with cysteine ​​using the Liang method. The ratio of cysteine ​​to gold nanoparticles was 51:1. After stirring for 25 h, the mixture was centrifuged and washed 4 times, and then dispersed in ultrapure water to obtain Cys @ AuNPs. The concentration of the above gold nanoparticles in Cys @ AuNPs was 0.05 mg / mL. S3. A 3D mold of the tree frog's foot was constructed using SolidWorks. The mold had a hexagonal honeycomb structure with a side length of 650 μm, a thickness of 350 μm, and a spacing of 150 μm. The oxidized kudzu polysaccharide and type A porcine skin gelatin were dissolved in reverse osmosis water and then mixed thoroughly with the Cys@AuNPs obtained in step S2 to obtain a mixed solution. The concentration of oxidized kudzu polysaccharide in the mixed solution was 11 wt%, the concentration of type A porcine skin gelatin was 16 wt%, and the concentration of Cys@AuNPs was 1 mg / mL. The mixture was constructed using a 3D printer (Microarchs 140pro, SLA) with biocompatible resin, a printing precision of 10 μm, an exposure time of 5000 ms, and a single-layer thickness of 0.02 mm. The mixed solution was coated onto the tree frog's foot 3D mold, gelled at 38°C, and then cooled to 5°C. Removed at ℃, a conductive cardiac patch made of natural kudzu polysaccharide mimicking the foot of a tree frog was obtained.

[0118] A method for preparing a kudzu polysaccharide double crosslinked vascular stent coating includes the following steps: (1) Clean the stainless steel substrate. The stainless steel sheet has dimensions of 11 cm * 11 cm * 0.3 cm. It is ultrasonically cleaned in acetone and ethanol for 22 min each at 26 ℃. After cleaning, it is rinsed with ultrapure water and then subjected to ion beam-assisted plasma treatment. The stainless steel substrate is fixed on the sample holder. The cylindrical ion-assisted plasma deposition device (IAPP) made of stainless steel is connected to the radio frequency power supply (ENI RF). The frequency of the radio frequency power supply is 14 MHz. The substrate support is connected to the RUP-6 pulse generator (GBS Elektronik GmbH). The ion bombardment effect is enhanced by applying a negative bias voltage. The vacuum system of the IAPP device consists of an Ebara PDV250 mechanical pump and an Edwards NEXT400 turbomolecular pump. It is connected to the chamber through a butterfly valve. The precursor gas is introduced into the system through a shower head device. The IAPP device is connected to the Allicat Scientific mass flow controller. The sample holder is electrically connected to the grid 5 cm in front of it. After cleaning, the chamber is evacuated to 6 × 10 -5 Torr was then introduced, followed by the introduction of thin film deposition gas. Acetylene, nitrogen and argon were mixed and introduced into the system at constant flow rates of 6 sccm, 6 sccm and 21 sccm, respectively. After adjusting the working pressure of the chamber to 110 mTorr, 55W radio frequency power and 550V negative bias were applied. Deposition lasted for 5 min. The material was then ultrasonically cleaned in ethanol and single-distilled water and dried in a vacuum drying oven at 38 ℃ for 3 days to obtain plasma-modified stainless steel. (2) The above-mentioned double-bonded kudzu polysaccharide and thiolated kudzu polysaccharide were dissolved in reverse osmosis water to obtain a mixed solution. The concentration of double-bonded kudzu polysaccharide in the mixed solution was 6 wt%, and the concentration of thiolated kudzu polysaccharide in the mixed solution was 6 wt%. Then, the plasma-modified stainless steel obtained in step S1 was immersed in the mixed solution and reacted at 38 ℃ for 25 h. After that, it was washed with single-distilled water and dried with nitrogen to obtain a kudzu polysaccharide double crosslinked vascular stent coating.

[0119] Comparative Example 1 A hydrogel patch, compared to the tree frog-foot-inspired natural kudzu polysaccharide conductive cardiac patch prepared in Example 1, differs in that Example 1 does not contain gold nanoparticles. (GPS) Comparative Example 2 A double-bonded kudzu polysaccharide monocrosslinked hydrogel coating, compared to the kudzu polysaccharide double-crosslinked vascular stent coating prepared in Example 1, differs in that Comparative Example 2 does not contain thiolized kudzu polysaccharide (PSMA). Comparative Example 3 A thiolized kudzu polysaccharide monocrosslinked hydrogel coating, compared to the kudzu polysaccharide double crosslinked vascular stent coating prepared in Example 1, differs in that Comparative Example 3 does not contain double-bonded kudzu polysaccharide. (PSSH) Experimental Example 1 (1) The rheological properties of the tree frog-inspired natural kudzu polysaccharide conductive cardiac patches prepared in Examples 1-3 were tested. The hydrogel patch prepared in Comparative Example 1 was used as a control. The storage modulus (G') and loss modulus (G”) of the hydrogels loaded with different contents of gold nanoparticles (Au NPs) were tested using a Haake Mars 60 rheometer. The results are as follows: Figure 1 As shown, Figure 1 In the figures, 0.1 represents the result of the conductive heart patch made of natural kudzu polysaccharide mimicking tree frog feet prepared in Example 1, 0.05 represents the result of the conductive heart patch made of natural kudzu polysaccharide mimicking tree frog feet prepared in Example 3, 0.01 represents the result of the conductive heart patch made of natural kudzu polysaccharide mimicking tree frog feet prepared in Example 2, and GPS represents the result of the hydrogel patch prepared in Comparative Example 1.

[0120] Depend on Figure 1 It can be seen that as the content of gold nanoparticles increases, the storage modulus gradually increases, which means that the hydrogel has an enhanced ability to recover its original shape after being deformed, and has better mechanical strength and resistance to external forces.

[0121] (2) Next, the healing ability of the tree frog-like natural kudzu polysaccharide conductive cardiac patch prepared in Example 1 under different shear stresses was characterized using a rheometer. Step strain was tested on a cylindrical hydrogel sheet with a diameter of 12 mm and a height of 5 mm on a rheometer, with alternating strains of 1% and 300% shear force, a temperature of 37 ℃, a single cycle of 10 min, and a fixed frequency of 1 Hz. The results are as follows. Figure 2 As shown.

[0122] Depend on Figure 2 It can be seen that, under dynamic loading conditions, the conductive heart patch made of natural kudzu polysaccharide and mimicking the foot of the tree frog can dissipate energy more effectively and exhibit better durability and fatigue resistance when subjected to step strain tests ranging from 1% to 300%.

[0123] Experimental Example 2 Tensile tests were performed using a universal testing machine at a loading rate of 2 mm / min. A 3 mm thick hydrogel was cut into strips (25 mm long, 10 mm wide). The gauge length between the fixtures was 5 mm. The formula for calculating the tensile stress (δ) is:

[0124] Where F represents the maximum load force and A represents the cross-sectional area.

[0125] The draw ratio (λ) is the ratio of the increase in length after stretching to the original length, and is calculated using the following formula:

[0126] Where l is the length of the material after stretching, and l0 is the original length of the material.

[0127] The tensile stress-strain curves of the tree frog-inspired natural kudzu polysaccharide conductive cardiac patches prepared in Examples 1-3 are shown below. Figure 3 As shown, Figure 3 In the figures, 0.1 represents the result of the conductive heart patch made of natural kudzu polysaccharide mimicking tree frog feet prepared in Example 1, 0.05 represents the result of the conductive heart patch made of natural kudzu polysaccharide mimicking tree frog feet prepared in Example 3, 0.01 represents the result of the conductive heart patch made of natural kudzu polysaccharide mimicking tree frog feet prepared in Example 2, and GPS represents the result of the hydrogel patch prepared in Comparative Example 1.

[0128] Depend on Figure 3 It can be seen that with the increase of gold nanoparticle content, the tensile strength and elongation of the hydrogel are significantly improved. The average elongation is 110%, 180%, 480%, and 650%, respectively. The average tensile strength is 303, 487, 1700, and 2292 kPa, respectively, with an increase of 750%.

[0129] Experimental Example 3 The anti-Gram-positive bacteria performance of the biomimetic tree frog foot-like natural kudzu polysaccharide conductive cardiac patches prepared in Examples 1-3 was tested using the inhibition zone method and co-culture method. The colony maps, inhibition zone sizes, and bacterial viability / death staining results after co-culturing Staphylococcus aureus with different patches for 12 hours are shown below. Figure 4 As shown. Figure 4 In the table, each row from top to bottom shows the results of the conductive cardiac patch's anti-Gram-positive bacteria performance using the inhibition zone method, the anti-Gram-positive bacteria performance using the co-culture method, and the bacterial live / dead staining results. Each column from left to right shows the results of the blank control, the conductive cardiac patch made with the mimicking tree frog's foot natural kudzu polysaccharide prepared in Example 2 (0.01), the conductive cardiac patch made with the mimicking tree frog's foot natural kudzu polysaccharide prepared in Example 3 (0.05), and the conductive cardiac patch made with the mimicking tree frog's foot natural kudzu polysaccharide prepared in Example 1 (0.1).

[0130] Depend on Figure 4 It is known that the biomimetic tree frog foot-like natural kudzu polysaccharide conductive cardiac patch provided by the present invention has good antibacterial properties.

[0131] Experimental Example 3 To investigate the effect of a conductive cardiac patch made from natural kudzu polysaccharide, mimicking the foot of a tree frog, on cardiomyocyte viability under a hemodynamic / reverse (H / R) model, cardiomyocytes were divided into four groups and treated as follows: (1) control: untreated cells; (2) positive: cells under the H / R model; (3) GPS: cells treated with the hydrogel patch prepared in Comparative Example 1 under the H / R model; (4) GPS@NPs: cells treated with the conductive cardiac patch made from natural kudzu polysaccharide, mimicking the foot of a tree frog, prepared in Example 1 under the H / R model.

[0132] The above four groups of cardiomyocytes were stained for CX43 staining: After cell fixation, the cells were permeabilized, 5% BSA blocking solution was added, and the cells were incubated with primary antibody overnight. Then, FITC-labeled secondary antibody was added, and the cells were incubated at 37 ℃ in the dark for 1 hour. The fluorescence image of CX43 protein expression in cardiomyocytes is shown below. Figure 5 As shown, Figure 5 In the image, the magnification of the first row is 40, and the magnification of the second row is 100. The semi-quantitative fluorescence analysis results of CX43 are as follows: Figure 6 As shown.

[0133] Depend on Figure 5 and Figure 6 It can be seen that the expression level of CX43 in the GPS @Au NPs group was 4 times higher than that in the model group (P<0.001). This may mean that the conductive cardiac patch of natural kudzu polysaccharide mimicking tree frog feet enhances the electrophysiological and metabolic coupling between cardiomyocytes. This enhanced electrical coupling helps the synchronous electrical signal transmission between cardiomyocytes, maintains the normal rhythmic contraction of the heart, and helps the heart regeneration and repair.

[0134] Test Example 4 To establish a mouse model of myocardial infarction (MI): (1) Forty-five male SPF-grade C57 mice aged 3-4 months were randomly selected, anesthetized with isoflurane, their chest hair was shaved and disinfected, and placed on a cardiac operating table. The ventilator parameters were: inspiratory-to-expiratory ratio of 1:1; frequency of 70 breaths / min; and tidal volume of 0.35 mL. (2) A vertical incision was made in the middle of the rat's neck. The fascia and muscles were bluntly separated with surgical forceps. The sternum was carefully cut open with surgical scissors, and the heart was exposed by squeezing. (3) The proximal end of the left coronary artery (LAD) was identified and ligated using the left anterior descending artery ligation method. Subsequently, the downstream myocardial region was ischemic, the ventricular muscle turned cyanotic, the ventricular contraction weakened, and ST segment elevation was observed on the electrocardiogram. This indicated that the rat model of myocardial infarction was successfully established. The control group did not ligate after needle insertion at the above site and served as a sham surgery model.

[0135] Mice with myocardial infarction (MI) were treated by dividing them into four groups and administering the following treatments: (1) Sham group: untreated mice; (2) MI group: mice with acute myocardial infarction model; (3) GPS group: mice with hydrogel patches prepared in Comparative Example 1 applied to the infarct area under myocardial infarction model; (4) GPS@Au NPs group: mice with conductive cardiac patches made in Example 1, mimicking the foot of a tree frog, applied to the infarct area under myocardial infarction model. 30D in vivo echocardiograms of the four groups of mice are shown below. Figure 7 As shown.

[0136] Depend on Figure 7 It was observed that the MI group exhibited typical features of myocardial infarction, with a marked decrease in cardiac contractility, manifested as a significant reduction in ejection fraction (EF) and fractional shortening (FS). Left ventricular end-diastolic diameter (LVIDd) and end-systolic diameter (LVIDs) increased by 43.3% ± 3.1% and 62.4 ± 5.9%, respectively (P<0.001). These changes indicate progressive deterioration of cardiac function in the MI group, consistent with the established pathological progression after myocardial infarction, where loss of myocardial tissue leads to decreased systolic function and subsequent ventricular wall remodeling. The hydrogel patch treatment group showed significant improvement in cardiac function. Compared with the MI group, the GPS group showed increases in EF and FS to 45.3±2.8% and 22.1±1.7%, respectively (P<0.05), while the improvement was even more significant in the GPS@Au NPs group (EF 73.7±3.2%, FS 67.6±2.3%, P<0.001). The ventricular remodeling index showed that the LVIDd and LVIDs of the GPS @ Au NPs group were reduced by 41.3% and 60.3% respectively compared with the MI group (P<0.001), indicating that implantation of the tree frog foot-inspired natural kudzu polysaccharide conductive cardiac patch provided by the present invention can inhibit pathological deterioration, prevent left ventricular enlargement and improve cardiac function.

[0137] Masson staining results of the heart tissues of the above four groups of mice after 28 days are as follows: Figure 8 As shown, the statistical results are as follows: Figure 9 As shown. Figure 9 In the middle, the left figure shows the proportion of myocardial infarction in mice, and the right figure shows the comparison results of the cross-sectional area of ​​the distal infarct region in different groups (n=3, ***p<0.001).

[0138] Depend on Figure 8 and Figure 9 It can be seen that the GPS@Au NPs group exhibits a significant advantage in improving myocardial structural remodeling: compared with other intervention groups, the gross specimens of the GPS@Au NPs hydrogel group showed that the ventricular geometry of the treatment group was maintained optimally, with the infarct area accounting for only 18.75±2.75% (vs 58.55±2.21% in the MI group, P<0.001), the boundary area was reduced, the left ventricular wall thickness was significantly increased, and the fibrosis area was significantly reduced. This indicates that the conductive cardiac patch inspired by the tree frog's foot and natural kudzu polysaccharide provided by this invention has excellent myocardial structural remodeling capabilities.

[0139] Experimental Example 5 SEM images of kudzu polysaccharide double cross-linked vascular stent coating after being gripped on a balloon and expanded, as shown below. Figure 10 As shown.

[0140] Depend on Figure 10 It is known that the kudzu polysaccharide double cross-linked vascular stent coating exhibits strong adhesive strength and is flexible enough to deform the stent during balloon expansion without cracking or peeling off from the stent strut, and can be used as a coating to prepare the surface of vascular stents.

[0141] Experimental Example 6 Leveraging the excellent encapsulation capabilities of the kudzu polysaccharide double-crosslinked vascular scaffold coating and the long-lasting autofluorescence properties of carbon dots, 100 μg / mL of carbon dots were mixed into the precursor solution of the kudzu polysaccharide double-crosslinked vascular scaffold coating. The carbon dots were incorporated into the coating during the free radical polymerization process, enabling the coating to be excited by a green light source. Subsequently, the stability and degradation process of the kudzu polysaccharide double-crosslinked vascular scaffold coating under strong blood flow conditions were simulated using flow chamber chip technology. A peristaltic pump was used to adjust the PBS flow rate to 10 times the physiological blood flow rate (51.21 ml / min). On days 0, 3, 7, and 12 of the experiment, the kudzu polysaccharide double-crosslinked vascular scaffold coating (PSMASH) prepared in Example 1, as well as the double-bonded kudzu polysaccharide single-crosslinked hydrogel coating (PSMA) and the thiolized kudzu polysaccharide single-crosslinked hydrogel coating (PSSH) prepared in Comparative Example 2, were scanned using a laser confocal microscope (CLSM). The results are as follows: Figure 11 As shown, the amount of coating retained is directly proportional to the fluorescence intensity.

[0142] Depend on Figure 11It is known that PSSH, due to its thinner thickness and lower crosslinking density, exhibits a significant decrease in fluorescence intensity after 3 days and a decrease in homogeneity after 7 days. In contrast, PAMASH possesses a dense and uniform film coating, a characteristic that allows it to remain in the flow chamber for up to 12 days and degrade stably.

[0143] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a conductive cardiac patch made of natural kudzu polysaccharide mimicking the foot of a tree frog, characterized in that, Includes the following steps: S1. Heat and stir the tetrachloroauric acid trihydrate solution to boiling, add sodium citrate solution, and after the reaction, gold nanoparticles are obtained. S2. The gold nanoparticles obtained in step S1 were modified with cysteine ​​using the Liang method, stirred and reacted, washed, and then dispersed in ultrapure water to obtain Cys@AuNPs; S3. Construct a 3D mold of the tree frog's foot. Dissolve oxidized kudzu polysaccharide and gel in reverse osmosis water, and then mix them evenly with Cys@AuNPs obtained in step S2 to obtain a mixed solution. Coat the mixed solution into the 3D mold of the tree frog's foot. After gelation, obtain a conductive heart patch with natural kudzu polysaccharide imitating the tree frog's foot. The preparation method of the oxidized kudzu polysaccharide includes the following steps: dissolving kudzu polysaccharide in reverse osmosis water, then adding boiling water, stirring and cooling, then adding sodium periodate solution dropwise, adding ethylene glycol after reaction, and finally dialyzing and freeze-drying to obtain oxidized kudzu polysaccharide.

2. The preparation method of the tree frog-inspired natural kudzu polysaccharide conductive cardiac patch as described in claim 1, characterized in that, In step S3, the concentration of Cys@AuNPs in the mixed solution is 0.01-0.1 mg / mL.

3. The preparation method of the tree frog-inspired natural kudzu polysaccharide conductive cardiac patch as described in claim 1, characterized in that, In step S3, the tree frog foot 3D mold has a regular hexagonal honeycomb structure with a side length of 550-650 μm, a thickness of 250-350 μm, and a spacing of 50-150 μm.

4. The application of the conductive cardiac patch made by the method of preparing the tree frog foot-like natural kudzu polysaccharide conductive cardiac patch according to claim 1 in the preparation of drugs for treating cardiovascular diseases.

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